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Imaging the elephant

In an ancient Indian tale, six blind men attempt to identify a large object by touch alone. One man believes he’s touching a pillar. Another is sure he senses a hanging rope. The others feel a bare wall, a snake, a spear, a cloth fan. Only when they combine their impressions is the object finally revealed to be an elephant.

Just as each blind man contributes knowledge of a unique feature of the pachyderm, different techniques can be combined to form a complete image of a biological molecule’s physical structure. One such technique, known as cryo-electron microscopy, or cryo-EM, is central to the work of several EMBL groups. Cryo-EM allows them to obtain high-resolution information about a molecule’s form, and, ultimately, its biological function.

In cryo-EM, as in all types of electron microscopy (EM), a beam of electrons is focused at the specimen. Some electrons pass straight through while others hit the specimen and are scattered. The sample’s unique pattern of interference with the electron beam reveals details of its structure.

Electron microscopy allows scientists to obtain high-resolution images because the wavelength of the electrons is very short; if the wavelength of EM electrons were represented by the diameter of a football, the wavelength of visible light would be about as long as 200 football pitches positioned goal-to-goal. Both EM and light microscopy are constantly advancing, but with current EM techniques scientists can see things that are over 1000 times smaller than what they can see with powerful light microscopy. Light microscopy can illuminate objects as small as bacteria, but some EM techniques can go as far as detecting individual atoms.

Cryo-EM is distinguished from other types of EM by its relatively less invasive method of sample preparation. In other kinds of EM samples are hardened and sliced into sections, or stained with heavy metals. But in cryo-EM, samples are preserved in or close to biological conditions. A sample is kept below freezing temperature (hence “cryo”), holding target structures in place and preserving their form.

Trying a Different Angle

Christiane Schaffitzel, an EMBL group leader in Grenoble, France, learned of the advantages of cryo-EM while studying ribosomes during her post-doctoral research at the Institute of Molecular Biology and Biophysics at ETH Zürich. She had been enlisting another structural technique – crystallography, whereby the three-dimensional properties of a molecule are obtained by analyzing a solid crystal made from many copies of the sample arranged in a regular lattice. But depending on the size or atomic interactions of a molecular complex, producing crystals can be a tricky business.

“My project was to crystallize a translating ribosome, but I never obtained crystals,” she says. “Still today, many years later, a ribosome with a nascent polypeptide in its tunnel has not been crystallized.” So Christiane asked Joachim Frank of Columbia University to examine the sample with cryo-EM. “It didn’t even take half a year. I realized this is actually a great method to solve complexes which are not very stable and which do not easily crystallize.”

Christiane has since found cryo-EM to be very useful for studying how newly-formed proteins are targeted to different locations in a cell. In recent research, she and her colleagues combined cryo-EM with data from other techniques to better understand this process, called co-translational targeting.

“It is like a puzzle,” she says, “You take your electron microscopy data, you take your biochemical evidence, cross-links, mutations, fluorescence measurements, crystallographic data, bits and pieces, and then…put it [all] together to generate a quasi-atomic model that makes sense.”

A Tool in the Toolbox

Christiane’s group employs single-particle cryo-EM, in which multiple copies of a molecule are imaged in two dimensions. A three-dimensional model can then be created with software that combines all the images, in effect taking an average structure of all the copies.

But cryo-EM could actually be represented by two blind men in the story of the elephant. In a second approach, called cryo-EM tomography, the sample is tilted in between multiple exposures to the electron beam. A three-dimensional image is created from one copy of the structure.

Group leader John Briggs uses cryo-EM tomography in his work at EMBL Heidelberg. He studies how proteins induce pockets of cellular membranes to bud off. The process is important both for the formation of vesicles – membrane-enclosed sacs that transport materials into, out of and within a cell – and for assembly of membrane-bound viruses like HIV.

“We’re interested in the fundamental mechanism,” John says. “How can you collect together a bunch of different proteins to turn a piece of the plasma membrane or some other cellular membrane into the membrane which surrounds a virus or a vesicle?”

To address the question, John, like Christiane, combines cryo-EM images with data collected through other means. These include other types of electron microscopy and fluorescence microscopy, a light microscopy technique that indicates the presence and position of specific proteins.

“We’ve been looking at the structures of various intermediate steps between the immature and the mature HIV virus to try to understand the structural changes that occur during the maturation process,” John says. “We use different types of microscopy at different stages of the project depending on what type of information we need at that particular time. We get complementary information from the different methods.”

John’s colleague Martin Beck also constructs cryo-EM tomography images of cellular structures. Martin, also group leader in Heidelberg, studies the structures of large biological molecules, with a focus on nuclear pore complexes. These multi-protein assemblies dot the membrane surrounding the nucleus of a cell and help transport materials in and out of the nucleus.

“We look at nuclear pores still embedded in the nuclear membrane,” Martin says. “We have determined the general structure of a nuclear pore, and now we are trying to figure out where certain modules of the complex are precisely localized.”

Martin combines cryo-EM with other techniques as he moves closer to a complete structural model of the nuclear pore complex. He hopes to one day chart the structure down to the level of its component atoms. Single-particle cryo-EM, crystallography, and mass spectrometry– a broadly informative technique that measures particle mass and electric charge – all help Martin uncover the intricacies of this “elephant” of a molecule.

The Titan Krios

Martin and John’s projects will soon be accelerated, thanks to the recent addition of the innovative Titan Krios to EMBL Heidelberg’s electron microscope collection. According to John, the instrument will allow for faster, more automated data collection.

“The Titan has been used in the materials sciences, and now it has been adapted for cryo,” says Carsten Sachse, another EMBL Heidelberg group leader who works with cryo-EM. “It has very good optics, and it has been shown to be quite a boost for biology in terms of resolution.”

Carsten will be using the Titan Krios to investigate how protein aggregates are cleared from a cell. Abnormal protein accumulation is characteristic of neurodegenerative illnesses, including Alzheimer’s, Parkinson’s, and Huntington’s diseases. By studying how protein aggregates are normally removed from a cell through endocytosis, or eliminated within a cell by autophagy, Carsten hopes to better understand diseases in which these mechanisms seem to fail.

“There are clear molecular links between the two processes,” Carsten says. “There’s a multitude of proteins involved that are thought to function like little machines inside the cells and somehow reshape membranes. We would like to understand more closely how they work.”

The Titan Krios will also benefit the research of other scientists. In one ongoing project, John and Carsten are working with EMBL group leader Marko Kaksonen on visualizing membrane budding in yeast cells. Meanwhile, Christiane is collaborating with EMBL Grenoble group leader Imre Berger, to determine the structure of major transcription machinery in eukaryotic cells.

These scientists continue a long tradition of cryo-EM breakthroughs at EMBL. In fact, early in EMBL’s history, group leader Jacques Dubochet and colleague Alasdair McDowall invented cryo-EM sample preparation. They published their method in 1981 in a brief, typewritten paper in the Journal of Microscopy, outlining a way of rapidly cooling a sample so that the liquid solution forms a glass-like layer, instead of freezing into crystals.

In the tradition of many important discoveries that are initially underappreciated, Jacques and Alasdair’s paper faced a prevailing belief that such “vitrification” of liquid water was thermodynamically impossible. Nonetheless, the validity of the method was soon evident, and formed the foundation of the cryo-EM techniques used today.

What’s next for cryo-EM?

“What we hope for the future of cryo-EM is that it will be a standard technique alongside crystallography and NMR [nuclear magnetic resonance spectroscopy, a technique that can identify the components of a molecule based on their magnetic properties],” Carsten says. “It’s just not as mature as other techniques, and it’s not a common thing that’s available at every university. Cryo-EM will be quite powerful one day.” Indeed, improving cryo-EM techniques was part of Carsten’s development as a scientist, and he and his colleagues continue to refine the method.

In the story of the blind men and the elephant, careful study by each man allowed for insight into a bigger picture. As it improves, cryo-EM – used in parallel with other techniques – will allow for increasingly detailed insights into complex biological structures. If the blind men had put similar efforts into their own powers of examination, they might have gone on to catalogue the entire animal kingdom.